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Summary

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Abstract

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

Use 2000-7000 cells per aggregate. Add 200 ul L-15 per aggregate into each tube.

Split cells (200 μl) into new eppendorf tubes.

Spin at 1000 rpm for 2 min at RT.

Incubate for 48 hrs at 27°C.

3. Implantation of Aggregrated Cells

Make a slit in the cornea of newt eye and remove the lens.

After lens removal, place the IPE cell aggregate just below the corneal tissue on the ventral side of the eye.

The newts are kept for a month to let them regenerate the lens.

4. Embedding of Newt Eye

Remove the newt eyes implanted with the aggregate and place it in phosphate buffered saline (PBS).

Fix in 4% paraformaldehyde at 4°C for at least 4 hrs or overnight.

Wash in PBS, 4°C for 30 minutes.

Treat it with 0.85% saline: 4°C, 30 minutes.

Wash in Saline/Ethanol (1:1): RT for 15 minutes.

Wash in 70% Ethanol: RT, 15 minutes. Repeat it once.

Wash in 85% Ethanol and 95% Ethanol at RT, 30 minutes each

Wash in 100% Ethanol: RT, 30 minutes. Repeat it once.

Store at 4°C or continue.

Wash in 100% xylene: RT, 30 minutes. Repeat it once.

Treat with xylene/paraffin (1:1): 60°C, 45 minutes.

Treat with 100% Paraffin: 60°C, 20 minutes. Repeat it two more times.

Embed in embedding molds.

5. Sectioning

Prepare 15 μm sections of the embedded eye using a microtome.

Place the eye sections on a gelatin coated slide and leave it on a slide warmer.

6. Staining

Place slides in xylene for 10 minutes. Repeat it once more.

Hydrate in: 100%, 95%, 80%, 70%, 30% ethanol for 1 minute each

Rinse in DI water for a minute.

Wash in PBS for 15 minutes.

Wash in PBST (0.2% Triton-X 100 in PBS) for 15 minutes.

Wash in PBS for 15 minutes.

Place in blocking solution (10% goat secondary antibody) for an hour.

Place in primary antibody for overnight.

Wash primary antibody in PBS for 15 minutes.

Wash in PBST for 15 minutes.

Wash in PBS for 15 minutes.

Place in secondary antibody for 2 hours in dark (1:100 dilution in PBS).

Wash in PBS, PBST, and PBS for 15 minutes each and then mount.

Observe the slides under a fluorescence microscope.

7. Representative Results:

This procedure of culturing newt iris cells has been utilized to study the regeneration potential of the dorsal and the ventral IPE cells. Moreover, it is also possible to study specific genes that contribute towards the lens regeneration mechanism in newt eye. When the cells have been cultured for 2 weeks (Figure 1) they can be transfected by genes to examine their function in lens regeneration. Of particular interest are genes that might induce the ventral iris. Since the ventral iris cells cannot transdifferentiate to lens (Figure 2) the inductive function of a candidate gene can be studied. In the past, using this technique we have shown that when six-3was over expressed in the presence of retinoic acid lens induction was observed from the ventral iris 6. In Figure 3we can see that the ventral iris aggregate gave rise to a fully grown and differentiated lens (arrowhead), not different than the host lens from the dorsal iris (arrow).

Figure 1. a) Dorsal iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. b) Ventral iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. Note that pigmentation persists to this stage in both dorsal and ventral iris cells.

Discussion

This protocol has established an in vitro system to study lens regeneration mechanisms in newts. Since the aggregates (either dorsal or ventral faithfully follow their in vivo behavior during regeneration this technique can alleviate the tremendous effort required for transgenesis in newts and can be used for gain of function as well as loss of function experiments 7,8,9 . Also, the aggregates or the irises as a whole can easily be treated with growth factors and examine their effects as described in this protocol. For example the role of BMP pathway has been studied using this technique 6.

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